PMID:23621611

Citation	Xie, WH, Pang, F, Niu, YF, Zhang, MH, Yang, WD, Liu, JS, Yan, DG and Li, HY Functional characterization of an ACCase subunit from the diatom Phaeodactylum tricornutum expressed in Escherichia coli. Biotechnol. Appl. Biochem. 60:330-5
Abstract	The marine diatom Phaeodactylum tricornutum, a widely used forage species, has a storage lipid content of up to 30% dry cell weight. To explore the mechanism behind the high storage lipid accumulation in this diatom, acetyl-CoA carboxylase (ACCase), which catalyzes the first committed step of the fatty acid biosynthetic pathway, was characterized in this study. A homogeneous type of ACCase (PtACC) was identified from P. tricornutum by homology searches. The first exon of the ACCase gene (PtACC-1) was cloned. PtACC-1 was fused with a Myc epitope tag and cloned into plasmid pMD18 driven by the LacZ promoter and expressed in Escherichia coli. The expression of the PtACC-1-Myc protein was verified by Western blot. The neutral lipid content in transformed E. coli increased substantially by twofold as determined by Nile red fluorescent dye staining. Concomitantly, ACCase activity increased by 1.72-fold. The fatty acid composition, analyzed by GC-MS, demonstrated a significant difference in the ratio of saturated fatty acids and monounsaturated fatty acids (MUFAs). MUFAs of PtACC-1 expressing cells increased by 13%. This study represents the first characterization of the key domains of ACCase from a diatom and demonstrates high neutral lipid accumulation in E. coli expressing PtACC-1, providing an additional genetic resource with the potential for biodiesel development.
Links	PubMed Online version:10.1002/bab.1091
Keywords	Acetyl-CoA Carboxylase; Cloning, Molecular/methods; Diatoms/genetics; Diatoms/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Exons/genetics; Fatty Acids/genetics; Fatty Acids/metabolism; Gene Expression/genetics; Phylogeny; Protein Structure, Tertiary/genetics; Protein Subunits/genetics; Protein Subunits/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
PHATC:B7G7S4		GO:0003989: acetyl-CoA carboxylase activity	ECO:0000315:		F		Figure 4 shows that when E. coli is transformed with the P. tricornutum ACCase subunit catalyzing the first committed step of the fatty acid biosynthetic pathway, the neutral lipid content significantly increases. When E. coli was transformed with the empty vector there was no neutral lipid content change compared to untransformed E. coli. Table 1 quantifies the ACCase enzyme activity, showing that the mutant E. coli containing P. tricornutum ACCase has significantly higher ACCase activity compared to the empty vector or the untransformed E. coli.	complete CACAO 9770
PHATC:B7G7S4	enables	GO:0003989: acetyl-CoA carboxylase activity	ECO:0000315: mutant phenotype evidence used in manual assertion		F		Seeded From UniProt	complete

References

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